1. Field of the Invention
The present invention relates to a gene coding for a thermostable DNA ligase from Aquifex pyrophilus, a hyperthermophilic Bacterium, a protein having an amino acid sequence expressed therefrom and a probe used for detecting said ligase DNA. More particularly, the present invention is directed to a gene having a base sequence of SEQ. ID. NO: 3 and a thermostable DNA ligase having an amino acid sequence expressed therefrom (SEQ. ID. NO: 4).
2. Description of the Prior Art
Recently, attempts have been made to develop an enzyme in a microorganism growing in a special environment or to improve an existing enzyme so as to maintain its activity even in a special environment. Specifically, a microorganism capable of growing at a temperature near or above 100xc2x0 C., the boiling point of water, was reported and an enzyme isolated from such an organism has been known to be able to maintain its activity without any degeneration at high temperatures near 100xc2x0 C. Therefore, an effort has been made to screen an industrially applied enzyme that is used at high temperatures from such a microorganism.
Microorganisms that grow at such a high temperature are referred to collectively as hyperthermophile. They have an optimum growing temperature of above 80xc2x0 C. and a type which grows at a temperature of above 115xc2x0 C. among them was reported (M. W. W. Adams, Annu. Rev. Microbiol. 47:627-658, 1993). So far, hyperthermophiles of more than about 50 to 60 types were reported and they are found primarily in hot marine sediments. Most hyperthermophiles are classified as a type of primitive bacteria or archaea, and thermotoga and aquifex are known as bacteria belonging to hyperthermophiles.
Aquifex pyrophilus belonging to eubacteria represents the deepest branching with the Bacteria in assay results due to comparison of 16s RNA sequence and was known as autotrophic bacteria, which obtains energy by oxidizing or reducing sulphur (Huber R. et al., Syst. Appl. Microbiol. 15: 349-351, 1992). Aquifex pyrophilus is a hyperthermophilic marine bacterium that grows at a temperature between 67xc2x0 C.-95xc2x0 C., with an optimum growing temperature of 85xc2x0 C. DNA ligase is an essential enzyme for a number of important cellular processes, including repair, replication and recombination of DNA in a cell since it catalyzes the formation of phosphodiester bonds at single-strand breaks in double-stranded DNA (Lehman I. R. Science, 186; 790-797, 1974, Lindahl, T. and Barnes, Annu. Rev. Biochem., 61: 251-281, 1992).
DNA ligase is present in all organisms, but its size, amino acid sequence and characteristics are very varied. DNA ligase is classified primarily into two types. One uses ATP as a cofactor and the other uses NAD+. All DNA ligases from eukaryotes and viruses require ATP, and sizes of DNA ligases range from 103 kDa for the human DNA ligase I to 41 kDa for the bacteriophage T7 enzyme. Eubacterial DNA ligases require AND+according to the facts that have been elucidated so far. Among the amino acid sequences of several bacterial DNA ligases that have been identified (Ishino, Y., et al., Mol. and Gen. Genet. 204: 1-7, 1986; and Barany, F., and Gelfand, D. H. Gene (Amst.) 109, 1-11, 1991), DNA ligases using NAD+as a cofactor are proteins of monomer form with a size of 70-80 kDa, show a high level of sequence homology between them and have little similarity to DNA ligases requiring ATP.
Thermostable DNA ligases are used as essential enzymes in various assays of genetic diseases. For example, mutations of a single base or specific base sequence can be detected by DNA ligase chain reaction (LCR), and genetic diseases caused by change of trinucleotide repeats can be detected by repeat expansion detection (RED). Useful applications of thermostable DNA ligases include the preparation of a primer that is capable of reading a sequence from six bases and the determination of the presence of localized DNA by circularizing oligodeoxynucleotide (Barany, F., Proc. Natl. Acad. Sci. USA 88, 189-193, 1991; and Nilsson, M., et al., Science 265; 2085-2088). Therefore, there is an increase in the demand to develop thermostable DNA ligases essential for a number of important cellular processes, including repair, replication and recombination of DNA.